More than 50% of human malignancies, including breast cancers, are associated with missense mutations or deletions of p53, and most of the missense mutations map to the DNA-binding domain of the protein. The nucleotide sequence of the human p53 gene and the amino acid sequence of the encoded p53 protein have been reported (Zakut-Houri et al. (1985), EMBO J., 4: 1251-1255; GenBank Code Hsp53). The p53 protein consists of 393 amino acids and its functional domains have been characterized (e.g., U.S. Pat. No. 6,326,464).
p53 is a sequence-specific transcriptional factor that transactivates a number of genes whose products are involved in cell growth regulation. These include WAF1/p21/Cip1, which arrests the cell cycle, GADD45 for DNA repair, and Bax and Fas/APO-1 to modulate apoptosis. Apoptosis is a complex process regulated by several pathways, some of which involve members of the Bcl-2 family and the Fas pathway. Wild-type p53 forms a tetramer to perform its tumor suppressor activity.
The sequence-specific DNA-binding activity of p53 appears to be negatively regulated by its C-terminal 30-amino acid (aa) segment (aa 363-393) and also by N-terminal proline-rich motifs located between aa 80-93. Synthetic peptides corresponding to the C-terminal domain of p53 such as residues aa 363-393 bind directly in vitro to over-expressed wild-type and mutant p53. Binding experiments with p53 proteins that contain selected deletions indicate that binding of the free aa 363-393 peptide to p53 requires the presence of both C-terminal aa 363-393 and N-terminal aa 80-93 sequences in the p53 protein. This observation suggests either that the free peptide may interact simultaneously with both regions or that the absence of either or both of these segments in p53 results in structural changes in the protein, lowering its affinity for the free peptide.
Deletion of either or both of these regulatory regions, as well as various C-terminal modifications, stimulate specific DNA binding of p53 in vitro. Previous studies demonstrate that the addition of a chemically modified C-terminal p53 peptide restored in vitro sequence-specific DNA binding function to mutant p53-273 (Arg to His). Furthermore, intranuclear microinjection of this peptide into SW480 colon carcinoma cells carrying an endogenous p53-273 His mutation restored transcriptional activation of a p53-responsive reporter construct.
It was previously demonstrated that a synthetic peptide derived from the C-terminal regulatory region of the p53 tumor suppressor protein (amino acids 361-382) could induce p53-dependent, apoptotic cell death in the presence of mutant p53 with minimal effect on cells with normal levels of wild-type 53 (A. L. Kim et al., Conformational and molecular basis for induction of apoptosis by a C-terminal peptide in human cancer cells, J. Biol. Chem. (1999) 274:34924-34931). Since p53 mutations occur in over 50% of common human cancers, a therapy directed at specifically killing p53-mutant cells would have wide application.